1. Field of the Invention
The present invention relates to the synthesis of glycopeptides and uses thereof, and more particularly, to the novel synthesis of V3 glycopeptides and the use of same for in vivo immunogens that induce broadly neutralizing antibodies.
2. Background of the Related Art
The human immunodeficiency virus type 1 (HIV-1) is the retrovirus that caused the global epidemic of AIDS. Today, more than 40 million people are estimated to live with HIV/AIDS and the epidemic is still expanding [12]. There is no doubt that the best hope to stop the worldwide epidemic is an effective HIV-1 vaccine. To achieve a maximal protection, an effective HIV-1 vaccine may need to induce both humoral and cellular immunity [1, 2, 13-16]. While cytotoxic T lymphocytes (CTL) response is important to control and reduce HIV-1 infection by killing the infected cells [15], passive immunization experiments in various animal models have repeatedly demonstrated that neutralizing antibodies with appropriate specificity, when present in sufficient concentrations, can provide sterilizing immunity [17-21]. Therefore, the design of an immunogen that is able to induce broadly neutralizing antibodies remains a major goal in HIV-1 vaccine development.
The past two decades have witnessed tremendous advances in the understanding of the structure and function of the envelope glycoproteins in connection with their immunological properties [1, 3, 22, 23]. So far, a panel of neutralizing antibodies has been identified that are broadly reactive against HIV-1 primary isolates. These include monoclonal antibodies (mAbs) b12 and 2G12 that target discontinuous epitopes on gp120 [24, 25], and mAbs 2F5 and 4E10 that may target the membrane-proximal region of gp41 ectodomain [26-28]. The third variable region (V3) of gp120 is a “principal neutralizing determinant (PND).” Although major V3-specific antibodies are isolate-specific and neutralize only T-cell line adapted viruses or limit primary isolates, recently studies have also shown that some V3-specific monoclonal antibodies are able to neutralize primary isolates across clades [2, 29]. It becomes clear that these broadly neutralizing antibodies are unusual in that they are recognize either discontinuous epitopes or special conformational epitopes.
The hypervariable region (the V3 domain) of the envelope glycoprotein gp120 is highly immunogenic and was once considered “the principal neutralizing determinant (PND).” However, the V3 domain has been a controversial target for HIV-1 vaccine design mainly because of the highly variable nature of the sequence [1, 2, 85]. As a result, most V3-specific antibodies from the sera of early-infected patients or immunized animals are isolate-specific and neutralize only T-cell line adapted viruses or a very narrow range of primary isolates. However, recent data demonstrated that broadly reactive anti-V3 antibodies did exist, and some V3-specific poly- and monoclonal antibodies were able to neutralize a range of HIV-1 primary isolates across clades [86-90]. In addition, the V3 domain is accessible on native virus envelope [91].
The oligosaccharide components of glycoproteins have been implicated to play important roles in modulating protein's folding, stability, immunogenicity, and various cellular activities [30-33]. HIV-1 has two envelope glycoproteins, gp120 and gp41. They form trimeric complexes of heterodimers on the viral surface. Both are significantly glycosylated. The outer envelope glycoprotein gp120 carries about 24 N-glycans and the carbohydrates constitute about half of the molecular weight of gp120 [34-36]. The transmembrane glycoprotein gp41 carries 4 conserved N-glycans and the carbohydrates make 20-30% of its molecular mass [37, 38]. There are three major types of N-glycans in N-linked glycoproteins, namely, the high-mannose type, the complex type, and the hybrid type [39]. In the case of HIV-1 gp120, the nature (type) of N-glycans on individual glycosylation sites for some HIV-1 strains have been elucidated [34-36]. An important observation is that, by alignment, corresponding N-glycosylation sites among different HIV-1 strains seem to carry the same type of N-glycans [34-36].
Many studies have implicated that glycosylation affects the local or global conformations of peptides and proteins [8, 53-55]. For example, glycosylation usually stabilizes local conformations and induce turn-like structures of a polypeptide chain [53, 55, 56]. Experiments have demonstrated that not only the size, but also the nature and linkage type of the attached sugar chain, would have an impact on the underneath polypeptide conformations [57-61]. According to the carbohydrate analysis, the V3 domain of gp120 carries three conserved N-glycans within or adjacent to the loop, one complex type at N301, and two high-mannose type N-glycans at the N295 and N332 positions (HXB2 numbering), respectively [34-36]. Therefore, it is conceivable to think that individual N-glycans within or adjacent to the V3 domain will certainly influence the domain's conformations. This will, in turn, affect the antigenicity and immunogenicity of the V3 domain, particularly when a conformational epitope is involved. However, gp120 itself is too heterogeneous to be used for elucidating the detailed effects of glycosylation on local conformations of the V3 domain, even if site-specific mutation can selectively remove individual N-glycans within or adjacent to the V3 loop. For example, a typical gp120 has about 24 N-glycans but each N-glycan may exist in several different isoforms. As a result, over 100 glycoforms for a recombinant gp120 would exist [31]. Such heterogeneity in structure makes it extremely difficult to decipher the structure-function relationship of a given glycoprotein, and may sometimes yield confusing information as in the case of the immunogenicity of gp120 glycosylation mutants [40, 49]. To have a clear understanding of the roles that carbohydrates play in a glycoprotein and, particularly, to explore HIV-1 glycopeptides as novel immunogens, homogeneous materials are required. Synthesis seems to be the only practical means to provide various homogeneous glycopeptides for subsequent structural and biological studies.
While glycopeptides containing monosaccharides or a small oligosaccharide moiety can be prepared by conventional solid-phase peptide synthesis using glyco-amino acid derivatives as building blocks [110-112], the construction of large, biologically relevant glycopeptides carrying native N-glycans is still a challenging task [110, 113, 114], mainly because oligosaccharide chains, if pre-attached during solid-phase peptide synthesis, are susceptible to the acidic conditions used for peptide deprotection and cleavage from the solid support. On the other hand, no general chemical method is available to attach a sugar chain to a pre-assembled free polypeptide in a site-specific manner to form a full-size natural glycopeptide. To solve the problem, the current inventors and others have been exploring a novel chemoenzymatic approach using an endo-β-N-acetylglucosaminidase (ENGase) for adding an oligosaccharide to a pre-assembled polypeptide [115, 116]. Endo-β-N-acetylglucosaminidases (ENGases) are inherently a class of hydrolytic enzymes, but some possess significant transglycosylation activity and are able to transfer a N-glycan to a N-acetylglucosamine (GlcNAc) moiety in a GlcNAc-peptide acceptor to form a new glycopeptide. Endo-A from Arthrobacter can transfer a high-mannose type N-glycan to a GlcNAc-containing peptide [117], while Endo-M isolated from Mucor hiemalis prefers complex type N-glycan in transglycosylation [60, 118, 119]. Therefore, the distinct substrate specificity of the two endoglycosidases in transglycosylation will allow the construction of different glycoforms of glycopeptides.
The chemoenzymatic approach consists of two key steps: solid-phase peptide synthesis to prepare a GlcNAc-containing peptide precursor and the Endo-A or Endo-M catalyzed transfer of the N-glycan to the acceptor to accomplish the synthesis of the target glycopeptides.
Although the chemoenzymatic method allows a quick assembling of large glycopeptides, it still suffers with some weakness, such as the relatively low transglycosylation yield (generally 10-20%), the product hydrolysis, and the limitations of using only natural N-glycans as the donor substrates. Thus, it would be advantageous to develop a synthesis method to create synthetic homogeneous glycopeptides with increased transglycosylation without the limitation of using only natural N-glycans, wherein the synthetic homogenous glycopeptides may be used for conformational studies and for use as immunogens that induce broadly neutralizing antibodies.